Leukocyte dna viscosity test



United States Patent 3,515,517 LEUKOCYTE DNA VISCOSITY TEST H. A. Carper, Pullman, Wasl1., assignor to Uni-Tech Manufacturing Co., Sun Valley, Calif., a corporation of California No Drawing. Filed Jan. 31, 1967, Ser. No. 612,788 Int. Cl. G01m 11/08, 11/00, 31/16 US. Cl. 23-230 ll] Claims ABSTRACT OF THE DISCLOSURE A method and an improved reagent for determining leukocyte numbers in body fluids. The estimation of blood leukocyte numbers is accomplished by an analytical deoxyribonucleic acid viscosity test. A lysing reagent is prepared by making a solution of sodium lauryl sulfate in aqueous disodium ethylenediaminotetraacetic acid. The analytical method comprises the steps of preparing a predetermined quantity of aliphatic sulfate reagent, adding a quantity of body fluid containing leukocytes to the reagent, mixing the reagent and the body fluid, and measuring the viscosity of the reagent-body fluid mixture. The viscosity of the unknown solution is compared to viscosities on standard calibration curves.

BACKGROUND OF THE INVENTION Field of the invention This invention relates to the analytical determination of leukocyte numbers in body fluids. More specifically, this invention relates to a method and to an improved reagent composition for analytically determining leukocyte numbers in body fluids by measuring a change in viscosity of said body fluids.

Description of the prior art The estimation of blood leukocyte numbers can be accomplished by an analytical deoxyribonucleic acid viscosity test. One prior art method employs a specially designed glass funnel having a bore of such diameter that a reagent flow rate through the funnel can be precisely measured. The funnel is then used to measure the increase in viscosity produced by the interaction of leukr cyte DNA and alkylarylsulfonate (sometimes referred to as CMT reagent). The prior art test has several disadvantages. After addition of body fluids containing leukocytes to a tube containing CMT reagent, the reagent and the fluids must be mixed by inverting the tube exactly five times. There is little correlation between duplicate samples when the degree of mixing is increased. The total contact time of the body fluid and the reagent must not exceed 30 seconds prior to the determination of the capillary flow time; longer periods of contact result in poor correlation. The prior art leukocyte DNA viscosity test because of the above mentioned disadvantages is a cumbersome procedure that is extremely time sensitive and gives erroneous results if exact mixing proceures are not followed. Because of these disadvantages large numbers of samples cannot be tested at the same time.

Accordingly, it is an object of this invention to provide a new and improved viscosity test for determining leukocyte numbers in body fluids.

Patented June 2, 1970 It is another object of this invention to provide a test for analytically determining leukocyte numbers in body fluids, the test results being independent of mixing procedures and independent of contact time between the reagents and body fluids.

Yet another object of the present invention is to provide a new reagent composition for the analytical determination of leukocyte numbers in body fluids and particularly in blood.

SUMMARY OF THE INVENTION In one of its broadest aspects, the method of the present invention comprises the steps of preparing a predetermined quantity of aliphatic sulfate reagent, adding a quantity of body fluid containing leukocytes to the reagent, mixing the reagent and the body fluid, and measuring the viscosity of the reagent-body fluid mixture. The reagent comprises a solution of sodium lauryl sulfate in acqueous disodium ethylenediaminotetraacetic acid.

The method of the present invention and the new reagent composition used in connection therewith offers several significant advantages over the prior art methods. The present analytical procedure gives highly accurate results independent of the contact time between the reagent and the test solution, and independent of mixing procedure (so long as thorough mixing is accomplished). Thus, it is possible to test a large number of different samples without having to process them all at once (to prevent excessive contact between the reagent and the body fluid) and without having to give them exactly the same amount of mechanical mixing.

DESCRIPTION OF THE PREFERRED EMBODIMENT The invented method and the reagents used in such a method will be fully understood from the detailed description which follows, and which is illustrative of one presently preferred embodiment of the invention.

The analytical determination of leukocyte numbers in body fluids such as blood is frequently required in routine hematology. The method is based upon the viscosity increase produced by the interaction of leukocyte deoxyribonucleic acid (DNA) and sodium lauryl sulfate. The prior art test employed the interaction of deoxyribonucleic acid (DNA) and an alkylarylsulfonate. The latter reagent, however, has many disadvantages such as critical mix and contact times. The critical variables encountered with the alkylarylsulfonate are controlled when the sodium lauryl sulfate is substituted therefor. It is thought that inhibition of deoxyribonuclease is probably the manner in which this control is achieved. When the lysing action of the reagent ruptures the leukocyte and breaks the bond holding the deoxyribonucleic acid in a tight coil, it also apparently releases the deoxyribonuclease from the lysosomes. Deoxyribonuclease can rapidly lyse the long strands of deoxyribonucleic acid thereby reducing the viscosity of the solution. Sodium lauryl sulfate exerts some deoxyribonuclease inhibition, but the major inhibitory action of the reagent is supplied by the disodium ethylenediaminotetraacetic acid which binds the magnesium and manganese necessary for deoxyribonuclease activity.

An aliphatic sulfate having 12 carbon atoms in the aliphatic chain is used as the lysing agent in the present procedure. Specifically, sodium lauryl sulfate has been found to be a particularly effective reagent.

The sodium lauryl sulfate reagent is prepared by mixing approximately 3% by weight of sodium lauryl sulfate into a 1% disodium ethylenediaminotetraacetic acid solution. Additional lysing agents that could be used are related to the class of anionic sulfate surfactants derived from natural or synthetic fatty alcohols with a chain length of more than eight carbon atoms. The disodium ethylenediaminotetraacetic acid constituent of the reagent is important because it combines with magnesium and manganese ions and takes them out of solution. This in turn prevents the deoxyribonuclease from reacting with deoxyribonucleic acid and thereby reducing its viscosity. Other reagents that could be used for the lysing process are sodium or potassium salts of the aforementioned fatty alcohol-based anionics such as sodium octyl sulfate, sodium decyl sulfate, sodium tetradecyl sulfate, sodium hexadecyl sulfate and sodium cetyl sulfate. The disodium ethylenediaminotetraacetic acid is ultized in the reagent as a chelating agent; that is, it chemically binds metallic ions such as magnesium and manganese and removes them from solution. Consequently, the activity of deoxyribonuclease is greatly inhibited. Since deoxyribonuclease is responsible for breaking down and unravelling the deoxyribonculeic acid and thereby for decreasing the viscosity of a solution containing it, when the enzyme is inhibited by lack of magnesium and manganese ions the initial viscosity of a solution is retained because deoxyribonucleic acid is not broken down. It is possible to use other chelating agents in the reaction mixture such as sodium salts of nitrilotriacetic acid. Typically, the reagent can be prepared by adding 3% by weight of sodium lauryl sulfate to a 1% by weight aqueous solution of sodium ethylenediaminotetraacetic acid. Other proportions of reagents can, of course, be mixed within the range of 0.2 to 5.0% by weight of sodium lauryl sulfate. The sodium lauryl sulfate-disodium ethylenediaminotetraacetic acid solution is stable when stored at room temperature for many months.

Several aliquots of reagent sufficient to conduct a leukocyte number determination are pipetted into separate test tubes which are then stoppered. A convenient quantity of reagents is 3 milliliters which can be pipetted into 12x75 mm. test tubes.

The viscosity flow time of a reagent-body fluid mixture is determined by use of a specially designed funnel. The funnel will be described with respect to its use with a blood reagent mixture. It is, of course, possible to use the funnel to determine the leukocyte number of any body fluid containing leukocytes. The funnel employed to measure the viscosity produced by the interaction of leukocyte DNA and the sodium lauryl sulfate reagent can conveniently be made of glass and have a millimeter stem length and a 1.3 millimeter stern bore. Prior to determining the viscosity of any reagent-blood mixture the funnel must be calibrated to have a reagent flow time identical to that of the funnel from which calibration curves have been obtained. Thus, reagent-blood mixtures having known amounts of leukocytes therein are passed through a funnel and their flow time measured. From the measured flow time it is possible to determine the increase of viscosity of the solution and to correlate to the number of leukocytes in the blood. It is also necessary to measure the length of time for reagent without blood to flow through the funnel so that it is possible to calculate the flow time increase due only to leukocytes in the blood. The funnel used in the method of the present invention should have a reagent flow time identical to that of the funnel from which a calibration curve is obtained, otherwise no correlation can be obtained between flow time and leukocyte numbers. The reagent flow time of the funnel used in the present method is 5.6 seconds. This funnel allows correct determinations of total leukocyte count when using a standard calibration curve constructed from data obtained by measuring viscosity in a glass funnel having a reagent flow time of 5.6 seconds.

A predetermined quantity of blood or other body fluid containing leukocytes is next added to the test tubes containing sodium lauryl sulfate reagent. To insure the use of a uniform amount of blood in each viscosity test a glass capillary tube about 75 millimeters long with an inside diameter of about 1.2 millimeters can be employed. This tube is filled to capacity with blood or other body fluid which is then forced out of the capillary tube into the test tube containing sodium lauryl sulfate reagent.

The blood and reagent are mixed by mechanically agitating the test tube or other container. This agitation can be accomplished by inverting the test tubes at variable number of times until mixing has occurred. The bloodreagent mixture can then stand at room temperature until flow time measurements are to be taken. In contract to prior art methods the flow times measured when the present reagent is employed, are independent of both contact time and mixing procedure of the blood and the reagent.

After mixing the blood or other body fluid and the sodium lauryl sulfate solution, the mixture is transferred into a funnel as heretofore described. The amount of time taken for the blood-reagent mixture to traverse the stern of the funnel is precisely measured with a stop watch accurate to 0.1 second or other mechanical or electronic timing devices. The time interval so measured can then be related to the number of leukocytes in the blood by the use of a calibration curve which comprises the fiow times of blood samples having known numbers of leuko cytes. By comparing the flow time obtained in the present test to a similar flow time on the calibration chart, the number of leukocytes in the present blood sample can be obtained.

EXAMPLE The sodium lauryl sulfate reagent is prepared by adding 3 gms. of sodium lauryl sulfate to milliliters of an aqueous 1% disodium ethylenediaminotetraacetic acid solution. This solution is stable for many months.

Versenated or heparinized blood samples were obtained from dogs. (It should be noted that blood could be taken from any animal including man.) Total leukocyte counts in these blood samples as detremined by standard hemocytometer methods ranged from 5000 to 25,000 cells cu. mm.

Three milliliters of sodium lauryl sulfate reagent are pipetted into 12 x 75 mm. test tubes, capped with rubber stoppers and stored at room temperatures until used. To insure the use of a uniform amount of blood in each viscotity test 75 mm. glass capillary tubes with an inside diameter of 1.2 mm. can be employed. Each capillary tube can be filled to capacity with blood and expelled into a test tube containing sodium lauryl sulfate reagent. A small capillary bulb is used to force the blood from the capillary tube into the reagent.

The blood-reagent tubes are gently inverted a variable number of times and allowed to stand at room temperature for various lengths of time before determining the flow time of the blood-reagent mixture.

The glass funnel employed to determine the flow time of the blood reagent mixture has a 20 millimeter stem length and 1.3 millimeter stern bore. The same funnel is employed in each test and has a reagent flow time of 5.6 seconds.

The blood-reagent mixtures are each poured into the funnel and the time taken for the mixture to traverse the length of the funnel stem can be precisely measured with a stop watch accurate to 0.1 second. The flow times obtained for the various samples are compared with similar flow time on a calibration curve. The calibration curve is constructed by using blood samples in which the leukocyte number has been determined by standard hemocytometer techniques. The flow times are then measured by using a funnel which has a reagent flow time of 5.6 seconds. The curve is then constructed showing flow time versus leukocyte numbers.

Tables I and II show the flow times of the blood reagent mixtures each of which was handled in a different manner. There are negligible differences in flow times, thus indicating that the degree of mixing and the total contact time of the mixture is of little importance.

TABLE I Blood/SL3 reagent Blood/SL8 reagent contact time capillary flow time 1 minute 9.6 seconds 3 minutes 9.7 seconds 12 minutes 9.8 seconds 17 minutes 9.7 seconds 25 minutes 9.8 seconds TABLE II Immediately before Blood/SL8 reagent Number after adding transfer to capillary capillary flow time blood funnel (seconds) FIGURE 1 shows the viscosity flow times of various blood SLS reagent mixtures versus the total leukocyte count. Linear regression by the method of least squares allows calculation of the line y=0.423 X+ .14 where y: capillary flow time and X=total leukocyte count in thousands.

FIGURE 1 (Scatter plot and linear regression line) r la 1 i It is apparent from the foregoing discussion that the present method of determining leukocyte numbers in body fluids has many advantages over the prior art methods which use alkylarylsulfonates as the lysing reagent.

The present method can be used to determine leukocyte numbers in blood, milk and other body fluids with sufficient accuracy to make it a practical tool that can be used more easily than standard hemocytorneter methods. The primary advantage of the method of the present invention is that the flow times obtained are independent of the blood-reagent contact time and are also independent of the number of times the test tubes are inverted or amount of other mechanical agitation during mixing of the blood-reagent mixture. These advantageous factors make possible the testing of large numbers of samples at one time without the necessity of taking viscosity measurements on all of them within the same period. Thus, the blood samples collected earliest can be added to sodium lauryl sulfate reagent and allowed to stand any convenient length of time before flow time is measured. Furthermore, it is known that when the prior art reagents are used, prolonged agitation of the blood-reagent mixture results in the rapid disappearance of the diagnostic gel. The presently used sodium lauryl sulfate-EDTA reagent, however, stabilized the initial diagnostic gel primarily by inhibiting deoxyribonuclease activity.

Although this invention has been disclosed and illustrated with reference to particular applications, the principles involved are susceptible of numerous other applications which will be apparent to persons skilled in the art. The invention is, therefore, to be limited only as indicated by the scope of the appended claims.

What is claimed is:

1. A method of quantitatively determining the number of leukocytes in body fluids comprising the steps of:

reacting a sample of body fluid to be tested with a reagent mixture comprising a salt of an aliphatic sulfate and a chelating agent; and

determining the viscosity of the body fluid-reagent mixture.

2. The method of claim 1, wherein said aliphatic sulfate is one having from 8 to 10 carbon atoms and said chelating agent is a polyaminopolycarboxylic acid.

3. The method of claim 1, wherein said aliphatic sulfate is sodium lauryl sulfate and said chelating agent is disodium ethylenediaminotetraacetic acid.

4. The method of claim 1, wherein said reagent mixture comprises from about 0.2% to about 5.0% by weight of sodium lauryl sulfate in an aqueous solution containing about 1% by weight of disodium ethelendiaminotetraacetic acid.

5. The method of claim 1, wherein said reagent mixture comprises about 3% by weight of sodium lauryl sulfate in an aqueous solution containing about 1% by weight of disodium ethylenediaminotetraacetic acid.

6. The method of claim 1, wherein said aliphatic sulfate is taken from the group consisting of sodium octyl sulfate, potassium octyl sulfate, sodium decyl sulfate, potassium decyl sulfate, sodium tetradecyl sulfate, potassium tetra decyl sulfate, sodium hexadecyl sulfate, potassium hexadecyl sulfate, sodium cetyl sulfate and potassium cetyl sulfate.

7. A method of quantitatively determining the number of leukocytes in body fluids comprising the steps of:

mixing a sample of body fluid to be tested with a reagent solution comprising sodium lauryl sulfate and ethylenediaminotetraacetic acid, sufficient to completely react with said body fluid sample; and

measuring the flow time of the reagent-body fluid mixture, whereby the increase in viscosity of the body fluid due to reaction of the deoxyribonucleic acid with the reagent can be correlated to the number of leukocytes in the body fluids.

8. The method of claim 7, wherein the amount of reagent used can range from 2 milliliters to 4 milliliters when the sample of body fluid ranges from 0.05 milliliters to 1.0 milliliters.

9. The method of claim 2, wherein said flow rate is measured by allowing said body fluid-reagent mixture to gravity feed through an orifice of predetermined and constant dimension, and measuring the time taken to do so for a given quantity of mixture.

10. The method of claim 9, wherein the flow time of said body fluid-reagent mixture is measured by allowing said mixture to flow through a funnel with a stem having a bore of predetermined dimension sufliciently small to 1/1960 McCune 252137 X 8/1961 Schalm et a1. 23-230 OTHER REFERENCES Carrol, E. I. et al., Journal of Dairy Science, vol. 45, pp. 10947 (September 1962).

Schalrn, O. W. et al., Journal of the American Veterinary Medical Association, vol. 130, pp. 199-207 (March 1957).

Schalm, O. W. et 21]., Journal of the American Veterinary Medical Association, vol. 145, pp. 1177-83 (Dec. 15, 1964).

MORRIS O. WOLK, Primary Examiner E. A. KATZ, Assistant Examiner US. Cl. X.R. 4242 

